An exon-biased biophysical approach and NMR spectroscopy define the secondary structure of a conserved helical element within the HOTAIR long non-coding RNA
Graphical abstract
Introduction
Long noncoding RNA (lncRNA) are dynamic modulators of gene expression and can function as molecular scaffolds, associating with chromatin modifying complexes near genomic loci to influence chromatin structure and gene expression (Spitale et al., 2011, Rinn and Chang, 2012, Marchese et al., 2017). In terms of gene architecture and sequence similarity, most lncRNAs do not have identifiable homologs but there are thousands of human lncRNAs that have defined homologs and share similar expression levels in vertebrate genomes. Comparative transcriptome studies show that some lncRNAs exhibit a varying degree of sequence conservation across short nucleotide stretches that reside within exonic regions, suggesting that conserved functions require only short sequence or structural regions that can be tolerated within the syntenic architecture (Hezroni et al., 2015, Quinn et al., 2016, Ulitsky, 2016).
In addition, many homologous lncRNAs are alternatively spliced at levels approaching that of mRNAs and are thought to have rapidly evolved to acquire a functional importance (Schorderet and Duboule, 2011, Haerty and Ponting, 2015, Ulitsky, 2016). During evolution, alternative splicing and exon number globally increase, while exon length decreases (Koralewski and Krutovsky, 2011, Haerty and Ponting, 2015, Lin et al., 2016). This notion is also illustrated in the analysis of tertiary-quaternary structure in protein coding genes, where protein domain boundaries can correlate with exon boundaries (Richardson, 1981, Liu and Grigoriev, 2004). These generalities suggest that exon boundaries can stabilize or destabilize RNA elements and demarcate regions within large RNAs that contain a defined tertiary fold.
We sought to explore the correlation of gene sequence elements and domain boundaries within the context of lncRNAs and developed an RNA secondary structure determination strategy termed Exon-Biased structure probing. This method assumes that a secondary structure fold can be contained within an exonic region of the lncRNA. As an example, we analyzed the heterogeneity and folding properties of individual exons of the human HOX transcript antisense RNA (hHOTAIR) using native gel electrophoresis, thermal melting, and analytical ultracentrifugation. In addition, we interrogated the most stable and homogenously folded hHOTAIR exon in vitro using chemical probing and NMR spectroscopy.
HOTAIR is a classic example of a lncRNA involved in silencing specific homeotic genes in embryonic stem cells and whose overexpression is associated with tumor metastasis and poor prognosis (Rinn et al., 2007, Gupta et al., 2010). Human HOTAIR primarily contains six exons and was identified to function in trans, influencing the transcriptional repression of a distant chromosomal domain (Rinn et al., 2007, Gupta et al., 2010, Tsai et al., 2010). Based upon the UCSC genome browser, targeted RNA-sequencing of hHOTAIR has revealed the existence of at least six isoforms, with alternative splicing events generating an additional 16 different isoforms (Kent et al., 2002). Although the physiological regulation of hHOTAIR alternative splicing is unknown, it is possible that different splice isoforms could impact the tertiary fold of the RNA, potential RNA-protein interactions, and the extent of transcriptional repression.
We selected hHOTAIR using an exon-biased mapping approach because its RNA secondary structure has been extensively studied in vitro (Kertesz et al., 2010, He et al., 2011, Wu et al., 2013, Somarowthu et al., 2015, Portoso et al., 2017, Spokoini-Stern et al., 2020). The full-length hHOTAIR has been defined using multiple chemical probing strategies, identifying four large domains (Domain I-IV) that contain specific structured regions that are proposed to contain high sequence co-variation (Somarowthu et al., 2015). Domain I has the highest degree of covariation support when compared to other regions of hHOTAIR, yet some R-scape and power covariation studies suggest that hHOTAIR does not contain any evolutionarily conserved RNA structures (Rivas et al., 2017, Rivas et al., 2020). In this paper, we show that at least one region of hHOTAIR can adopt a homogenous tertiary fold and represents a structural domain that is preserved within an exonic boundary of hHOTAIR, suggestive of an evolutionarily conserved RNA structure. In addition, we propose that this exon-focused biophysical approach, when combined with hybrid structural bioinformatic studies, may serve as a generalizable strategy to examine the evolution of conserved lncRNA secondary structure within mammalian genomes.
Section snippets
HOTAIR RNA synthesis and purification
Double stranded (ds) DNA templates for each HOTAIR exon were generated from the pLZRS-HOTAIR plasmid (H. Chang, Addgene Plasmid ID #26110) (Gupta et al., 2010) by polymerase chain-reaction (PCR) with primer pairs as listed in Table S1. Forward primers contain a T7 RNA Polymerase binding site to initiate in vitro transcription reactions (Milligan et al., 1987). PCR reactions were purified via spin-column (Qiagen), eluted in RNAse-free water, and quantified by UV absorbance at 260 nm.
In vitro
Results
To probe the general interplay between splicing and the folding properties of lncRNAs, we sought to characterize the conformational heterogeneity of individual, isolated hHOTAIR exonic transcripts and identify potential regions that contain a highly stable secondary structure. HOTAIR exons 1 (60 nt) and 2 (126 nt) displayed significant heterogeneity, regardless of treatment (Fig. 1). In particular, exon 1 exhibited a marked propensity to dimerize in both non-denatured (‘native’) and refolded
Discussion
The characterization of functionally relevant lncRNA-mediated biological mechanisms requires multiple experimental biophysical and bioinformatic approaches that probe the structure and thermodynamic properties of the RNA target (Butcher and Pyle, 2011, Pyle, 2014, Chu et al., 2015, Yao et al., 2017, Jones and Sattler, 2019). Although there still remains limited and conflicting mechanistic insight into the structure–function relationships of HOTAIR (Tsai et al., 2010, Wu et al., 2013, Somarowthu
Conclusions
We combined an exon-biased method with chemical probing and NMR to determine that HOTAIR can form independent structural domains with varying degrees of heterogeneity and thermodynamic stability. Although this method may be applied to large ncRNAs with defined splice boundaries, the exon-biased structure determination of HOTAIR with NMR spectroscopy and chemical probing does validate that the conserved helix 10 serves as a core helical element within the 5′ region of HOTAIR (Somarowthu et al.,
CRediT authorship contribution statement
Ainur Abzhanova: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Alexander Hirschi: Conceptualization, Data curation, Formal analysis, Writing - original draft. Nicholas J. Reiter: Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing - original draft, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We would like to acknowledge the laboratory of Dr. Michael P. Stone for assistance with thermal melting experiments, Dr. Markus Voehler and the Vanderbilt University Center for Structural Biology, and Dr. Marco Tonelli and the National Magnetic Resonance Facility at Madison (NMRFAM). We thank Dr. William J Martin for comments and the laboratories of Drs. Manuel Ascano and Gregor Neuert (Vanderbilt University Medical Center) for discussions.
References (61)
- et al.
Applications of NMR to structure determination of RNAs large and small
Arch. Biochem. Biophys.
(2017) - et al.
Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation
Biophys. J.
(2006) - et al.
Native purification and analysis of long RNAs
Methods Enzymol.
(2015) - et al.
Noncoding RNAs: regulators of the mammalian transcription machinery
J. Mol. Biol.
(2016) - et al.
Principles of long noncoding RNA evolution derived from direct comparison of transcriptomes in 17 species
Cell Rep
(2015) - et al.
Advanced approaches for elucidating structures of large RNAs using NMR spectroscopy and complementary methods
Methods
(2020) - et al.
Effects of osmolytes on RNA secondary and tertiary structure stabilities and RNA-Mg2+ interactions
J. Mol. Biol.
(2007) - et al.
A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo
Blood
(2010) - et al.
Protein domains correlate strongly with exons in multiple eukaryotic genomes–evidence of exon shuffling?
Trends Genet
(2004) Looking at LncRNAs with the ribozyme toolkit
Mol. Cell
(2014)
The anatomy and taxonomy of protein structure
Adv. Protein Chem.
Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs
Cell
HOTAIR forms an intricate and modular secondary structure
Mol. Cell
Phylogenetic Analysis with Improved Parameters Reveals Conservation in lncRNA Structures
J. Mol. Biol.
Molecular mechanisms of long noncoding RNAs
Mol. Cell
Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)
Methods
Analysis of RNA folding by native polyacrylamide gel electrophoresis
Methods Enzymol.
RNAstructure: Web servers for RNA secondary structure prediction and analysis
Nucleic Acids Res
The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks
Acc. Chem. Res.
Nuclear magnetic resonance-assisted prediction of secondary structure for RNA: incorporation of direction-dependent chemical shift constraints
Biochemistry
Technologies to probe functions and mechanisms of long noncoding RNAs
Nat. Struct. Mol. Biol.
SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments
RNA
Ions and RNA folding
Annu. Rev. Biophys. Biomol. Struct.
NMR spectroscopy of RNA
ChemBioChem
Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis
Nature
Unexpected selection to retain high GC content and splicing enhancers within exons of multiexonic lncRNA loci
RNA
The sequence, structure and evolutionary features of HOTAIR in mammals
BMC Evol. Biol.
Challenges and perspectives for structural biology of lncRNAs-the example of the Xist lncRNA A-repeats
J. Mol. Cell Biol.
RNA structure. Structure of the HIV-1 RNA packaging signal
Science
The human genome browser at UCSC
Genome Res
Cited by (4)
G·U base pairing motifs in long non-coding RNAs
2023, BiochimieNMR of RNA - Structure and interactions
2023, Current Opinion in Structural BiologyEffect of targeted regulation of vasodilator activated phosphoprotein by IncRNA HOTAIR on cell migration and invasion of cervical cells
2023, Chinese Journal of Cancer Prevention and TreatmentThe multiple molecular dimensions of long noncoding RNAs that regulate gene expression and tumorigenesis
2022, Current Opinion in Oncology
- 1
Authors contributed equally to this work.
- 2
Present address: Mammoth Biosciences, 279 E Grand Ave., South San Francisco, CA 94080, United States.